Saliva plays a major role in maintaining oral health. Patients with decreased saliva secretion (symptomatically, xerostomia) exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease and microbial infections. Despite recent improvements in treating xerostomia, few scientific advancements have occurred which can be clinically applied toward restoration of compromised salivary gland function. Attempts have been made to advance restorative treatments (via development of an artificial salivary gland using a variety of extracellular matrices). Nonetheless, such restorative treatment models have proven incomplete due to poor differentiation and poor specification of mechanisms underlying secretion. We believe a Fibrin hydrogel (FH) scaffold (linked to growth factors and extracellular matrix proteins in growth factor-reduced-Matrigel, [GFR- MG]) will allow acinar cells to differentiate and make possible construction of an artificial salivary gland. We have tried two approaches to produce differentiated acinar structures (i.e., as evidence by high amylase protein expression), using GFR-MG and FH. GFR-MG allows acinar cells to from organized three- dimensional (3D) structures capable of developing tight junctions (TJ); however, the matrix itself is tumorogenic. Likewise, FH are safe, but cells grown on this scaffold do not form 3D acinar structures. Moreover, cells grown on either GFR-MG or FH alone do not reach full differentiation, so they cannot be used to build an acinar structure. Interestingly, whe these scaffolds are combined (GFR-MG/FH), many of the issues observed when cells are grown on GFR-MG or FH alone apparently are resolved. Specifically, a 3D acinar structure is formed (as was the case with GFR-MG alone) and amylase expression is increased. It remains a problem, however, that the hybrid matrix (GFR-MG/FH) still retains tumorogenic properties of GFR- MG and thus is not useful for growth of an implantable acinar structure. Because amylase production is a fundamental indicator of cell differentiation, we intend to investigate the molecular mechanisms that enhance amylase expression and consequent polarized apical secretion in acinar 3D constructs grown on GFR- MG/FH. Our studies will determine the optimal concentration of GFR-MG and FH for organization of acinar differentiated structures allowing single salivary acinar cells to organize into differentiated 3D structures (Aim 1). Additionally, w will determine and isolate the GFR-MG growth factors responsible for amylase expression in acinar cells (Aim 2). Finally, we will immobilize growth factors and extracellular matrix proteins into FH (by chemical conjugation and lentiviral gene delivery) to evaluate their effects on salivary acinar differentiation and secretory function (Aim 3). These studies should lead to better therapeutic strategies to restore salivary gland dysfunction that contributes to xerostomia in patients with compromised salivary function. PUBLIC HEALTH RELEVANCE: Proper salivary gland function is critical for oral health. Autoimmune disorders (such as Sjgren's syndrome), genetic diseases (such as ectodermal dysplasia), and irradiation therapies (for head and neck cancers) cause salivary gland dysfunction and lead to severe dryness of the oral cavity. This project aims to create functional salivary acinar structures (using modified fibrin hydrogels), which could contribute to the construction of a transplantable artificial salivary gland.